The neural crest is a transitory tissue of the vertebrate embryo which differentiates into a variety of cell types. By clonal analysis of crest cell differentiation I previously demonstrated the existence of pluripotent quail neural crest cells: single neural crest cells grown in isolation give rise to clones that contain melanocytes and adrenergic nerve cells. I also showed that melanogenesis and adrenergic differentiation can occur in the absence of non-crest cells, although extracellular matrix derived from surrounding non-crest cells appeared to play an important role in expression of the adrenergic phenotype. However, mechanisms that govern crest cell differentiation are still poorly understood. In order to study the regulation of crest cell differentiation in molecular terms, a) the crest cell progeny obtained in vitro has to be further characterized, and b) the cellular system has to be simplified. In order to achieve this goal, I propose to apply morphological and immunochemical methods in order to establish whether Schwann sheath cells are of neural crest origin and whether neuronal cells are present in the cultures which synthesize neurotransmitters other than catecholamines and acetylcholine, such as serotonin, substance P, somatostatin and enkephalins. With respect to simplification of the cellular system, I will devise a defined culture medium and establish permanent clonal cell lines by virus transformation of crest cells. An attempt to investigate the regulation of crest cell differentiation with the culture system presently available will include a) the establishment of the possible existence of neural crest stem cells with clonal studies, and b) manipulation of the differentiative fate of crest cell progeny with conditioned medium, tumor-promoting phorbol esters and ionophores.